Protein tyrosine kinases are a member of the eukaryotic protein kinase superfamily. Enzymes of this class specifically phosphorylate tyrosine residues of intracellular proteins and are important in mediating signal transduction in multicellular organisms. Protein tyrosine kinases occur as membrane-bound receptors, which participate in transmembrane signaling, or as intracellular proteins which take part in signal transduction within the cell, including signal transduction to the nucleus.
As such, phosphorylation of tyrosine by protein tyrosine kinases is an important mechanism for regulating intracellular events in response to environmental changes. A wide variety of cellular events, including cytokine responses, antigen-dependent immune responses, cellular transformation by RNA viruses, oncogenesis, regulation of the cell cycle and modification of cell morphology and phenotype are regulated by protein tyrosine kinases. Enhanced protein tyrosine kinase activity can lead to persistent stimulation by secreted growth factors which, in turn, can lead to proliferative diseases such as cancer, to nonmalignant proliferative disease such as arteriosclerosis, psoriasis and to inflammatory response such as septic shock. Decreased function can also be lead to disease. For example, a decrease in the activity of insulin receptor tyrosine kinase is a cause of various types of diabetes and severe reduction of the B cell progenitor kinase leads to human X-linked agammaglobulinemia.
Thus, there is a need for compounds which can modulate the expression of protein tyrosine kinases. Such compounds have utility in the treatment of diseases caused by over activity or underactivity of protein tyrosine kinases. Such compounds also have utility in studying the mode of action of protein tyrosine kinases and how these proteins regulate cellular functions and activities.
It has now been found that short peptides which are derivatives of the HJ loop of a number of different protein tyrosine kinases can significantly affect the activities of cells expressing the protein tyrosine kinase. xe2x80x9cHJ loopxe2x80x9d is defined hereinbelow. A number of examples are listed below:
Peptide derivatives of the HJ loop of c-Src Csk, c-Abl and c-Met have been found to inhibit the proliferation of a number of a different endothelial cell types (Examples 2 and 4).
Yet other peptide derivatives of the HJ loop of c-Src, Lck and endothelial cells"" tyrosine kinase receptors (Endoth) have been found to stimulate the proliferation of bovine capillary endothelial cells in vi tro (Example 2).
Peptide derivatives of the HJ loop of Lyn/Hck have been found to cause morphological changes in vascular smooth muscle cells in vitro (Example 5).
Specifically, cells incubated with Ac-IVTYGKI-NH2 (SEQ ID NO.: 1) and Ac-IVTYGRI-NH2 (SEQ ID NO.: 2), referred to as HJ24 and HJ32, respectively, become elongated and assume a spindle-like structure.
Peptide derivatives of the HJ loop of c-Src have been found to protect PC-12 neuronal cells in vitro from the effects of serum deprivation (Example 3).
Based on the aforementioned discoveries, novel peptides are disclosed herein which are peptide derivatives of the HJ loop of protein tyrosine kinases. Also disclosed are methods of identifying a peptide derivative of an HJ loop of a protein tyrosine kinase which modulates the activity of said protein tyrosine kinase. Methods of modulating the activity of a protein tyrosine kinase in a subject are also disclosed.
One embodiment of the present invention is a novel peptide which is a peptide derivative of the HJ loop of a protein tyrosine kinase. The peptide comprises between about five and about twenty amino acid residues or amino acid residue analogs and modulates the activity of the protein tyrosine kinase. The N-terminus and/or C-terminus of the peptide can be substituted or unsubstituted. The peptide can be linear or cyclic.
Another embodiment of the present invention is a method of modulating the activity of a protein tyrosine kinase in a subject. The method comprises administering a therapeutically effective amount of a peptide which is a derivative of an HJ loop of said protein tyrosine kinase, as described above.
Yet another embodiment of the present invention is a method of identifying a peptide which modulates the activity of a protein tyrosine kinase. The method comprises providing a xe2x80x9ctest peptidexe2x80x9d which has from about five to about twenty amino acids or amino acid analogs and which is a peptide derivative of the HJ loop of said protein tyrosine kinase. The test peptide is incubated with cells having a cellular activity or function under the control of said protein tyrosine kinase under conditions suitable for assessing the activity of the protein tyrosine kinase. The activity of the protein tyrosine kinase is assessed and compared with cells of the same cell type grown under the same conditions in the absence of the test peptide. A greater or lesser activity compared with cells grown in the absence of the test peptide indicates that the test peptide modulates activity of the protein tyrosine kinase.
The peptides of the present invention can be used in the treatment of a wide variety of diseases caused by overactivity and underactivity of a protein tyrosine kinase. Examples include, but are not limited to, cancer, diseases caused by proliferation of smooth muscle (e.g., restinosis and atherosclerosis) psoriasis, inflammatory disorders, diabetes, immune disorders and osteoporosis. The peptides of the present invention also have in vitro utilities, for example, in the generation of antibodies which specifically bind the protein tyrosine kinase whose HJ loop has a sequence or subsequence corresponding to the peptide. These antibodies can be used to identify cells expressing the protein tyrosine kinase and to study the intracellular distribution of the protein tyrosine kinase. In addition, the peptides can be used to identity and quantitate ligands which bind the HJ loop of the protein tyrosine kinase from which the peptide was derived.